The invention relates to mass spectrometry imaging of histologic thin tissue sections. The term “mass spectrometric image” of a thin tissue section which is obtained by mass spectrometry imaging (MSI) is defined here as an image which contains a mass spectrum with molecular information for every image point. The “mass spectrometric image” thus corresponds precisely to term “color image” which contains a color spectrum for every image point. The color spectrum contains the complete color information of the visible light spectrum, even if our eye summarizes the color spectrum into a single color impression. And just as it is possible to generate images of selected colors from a color image, for example red, yellow and blue images for a color print, it is possible to use a mass spectrometric image to generate “mass-selective images”, each of which displays the concentration of a molecular ion in its spatial distribution across the thin tissue section. Images which are derived from several selective images, and which can be used to spatially characterize the tissue states, are also of interest.
Histology is the science of human, animal and plant tissues, in particular of their structure and function. A histologic classification is generally carried out on a stained thin tissue section a few micrometers thick, and concerns the cell types present, the organ-specific differentiation of the tissue, bacterial and parasitic pathogens in the tissue, the disease states of the tissue and distributions of pharmaceutical products or their metabolites. The classification can be limited to one or more sub-areas of a tissue section or even apply to only one or more individual cells or organelles. The disease states of human tissue may relate to inflammatory diseases, metabolic diseases and the detection of tumors, especially the differentiation between benign and malignant forms of tumor or the prognosis of therapeutic success and survival expectation of a patient.
The generation of histologic tissue sections for an optical analysis involves the following steps: (a) the tissue is stabilized by deep freezing or chemical fixation, e.g. with formalin. (b) a thin section around 10 micrometers thick is cut with a microtome and (c) the tissue section is fixed, e.g. on a microscope slide, and stained.
Tissue stabilization means that the tissue structures, the cells of the tissue themselves and even intracellular structures (organelles such as the cell nucleus, endoplasmic reticulum, and mitochondria) are preserved in the tissue section. Usually, tissue stabilization is performed by deep-freezing. A well-known chemical tissue stabilization is termed “formalin-fixed paraffin-embedded” (FFPE); whereby the proteins within the tissue are cross-linked by reaction with formalin. Clinical archives hold millions of tissue samples, collected for more than a hundred years, stabilized by FFPE or similar methods. In routine histologic analyses, the structures of the tissue section are imaged with the aid of optical microscopes or with a “slide scanner”. A visual image of the tissue section recorded in this way can have a spatial resolution of about 250 nanometers.
The state of a tissue in relation to disease or infection with pathogens as compared to a healthy tissue sample can become apparent by a characteristic composition of substances. Usually, the substances are measured mass spectrometrically without imaging from homogenized pieces of tissue. The tissue state can be characterized by molecular information, in detail by the concentrations of different substances in relation to each other. If the substances are soluble and their concentrations sufficiently high, their concentrations can be detected by mass spectrometric analysis. The substances can be all types of biological substances, e.g. proteins, nucleic acids, lipids, polysaccharides or conjugates like glycoproteins or glycolipids. An unusual pattern can result when certain biological substances are modified, underexpressed or overexpressed. Proteins, in particular, can be modified in characteristic ways, e.g. by posttranslational modifications (PTM) or controlled degradation of the protein chain.
Mass spectrometry with ionization of the samples by matrix-assisted laser desorption and ionization (MALDI) has been used successfully for many years for the determination of molecular masses, and for the identification of biological substances, particularly proteins and peptides. This type of analytical technique can also be used for complex mixtures with some success. For example, methods of mathematical statistics can be used to mass spectrometrically determine the state of a tissue sample. Before these methods are used, a large number of tissue samples of different classifications (so-called “cohorts”) have to be provided, e.g. for the adjustment or learning of parameters. The sample formats can be homogenates of pieces of tissue or extracts.
In imaging mass spectrometric analysis, i.e. the acquisition of a mass spectrometric image, tissue sections are mass spectrometrically analyzed, usually with ionization by matrix-assisted laser desorption (MALDI). To this end, a thin-tissue section is placed onto an electrically conductive microscope slide as sample support. A thin layer of a matrix substance is then applied onto the tissue section by a suitable method not generating much lateral mixing of the tissue components, in such a way that finally the dried matrix substance layer contains the soluble peptides (and also other soluble substances) in an extracted form. The sample support is introduced into a mass spectrometer, and mass spectra of the individual image points are acquired.
The raster scan method according to Caprioli (U.S. Pat. No. 5,808,300 A) is predominantly used for the imaging mass spectrometric analysis; however, it is also possible to acquire a stigmatic image of a region of the tissue sample (Luxembourg et al., Analytical Chemistry, 76(18), 2004, 5339-5344: “High-Spatial Resolution Mass Spectrometric Imaging of Peptide and Protein Distributions on a Surface”).
In both cases, a “mass spectrometric image” of the tissue section is obtained, where for every image point the molecular information is present in the form of a mass spectrum. As is usual for MALDI, every mass spectrum is summed from a large number of individual spectra and covers an appropriate mass range, which can extend from around 100 to 60,000 atomic mass units. The region below 800 atomic mass units is measured to determine lipid distribution and the distribution of pharmaceutical products and their metabolites. The range between 800 and 60,000 atomic mass units is measured to determine the distribution of endogenous peptides and soluble proteins.
Various suitable methods for the preparation of tissue sections for mass spectrometry imaging analysis are known from the documents DE 10 2006 019 530 B4 and DE 10 2006 059 695 B3 (M. Schurenberg et al.; 2006). The matrix solution can be applied to the tissue section by pneumatic spraying, nebulizing by vibration, or by nanospotting of droplets, for example. It is no trivial task to apply the matrix solution because, firstly, a strong lateral diffusion of the biological substances must be avoided, secondly, the soluble biological substances must be extracted from the tissue section as completely as possible and incorporated into the crystals of the matrix layer, and thirdly, a favorable ratio of biologically relevant substances to impurities must be achieved. Some impurities greatly reduce the ionization yield. The kind of application of the matrix substance to the thin tissue section, the limitations for the spot diameter of the laser beam on the specimen, and also the quantities of substance required for the laser desorption mean that mass spectrometric images of tissue sections are currently limited to a spatial resolution of around 20 micrometers.
If the mass spectrometric images are acquired with a relatively coarse grid of 50 micrometers, this already produces 240,000 mass spectra for an area of 20 by 30 millimeters. Each time-of-flight mass spectrum can, in turn, comprise around 30,000 ion current measurement values or more. As is usual for MALDI, a hundred or more individual time-of-flight spectra are acquired and summed for each mass spectrum. Even in modern mass spectrometers with a high laser pulse rate, the acquisition of a mass spectrometric image takes many hours or even days depending on the size of the thin section and the width of the scanning raster selected.
One of the advantages of ionization by matrix-assisted laser desorption is that practically only singly charged ions of unfragmented analyte substance molecules are produced. It is therefore relatively easy to interpret the mass spectra. The mass spectra of the individual image points each show usually the mass signals of 20 to 400 soluble endogenous peptides in the mass range between 800 and 5,000 atomic mass units. The signals of the peptides emerge from a broad chemical background. Lighter proteins with less than around 5,000 atomic mass units are usually called peptides.
When MALDI time-of-flight mass spectrometers are used for imaging, a mass accuracy of around 50 millionths of the mass (50 ppm) can be achieved in the mass spectra of the image. If MALDI is used with other mass spectrometers, for example ion cyclotron resonance mass spectrometers or time-of-flight mass spectrometers with orthogonal ion injection, even better mass accuracies can be achieved.
“Monoisotopic ions” are defined as those ions from an isotopic group which are composed only of 1H, 12C, 14N, 16O, 31P, and 32S and contain no other isotopes of these elements. The monoisotopic ions are always the lightest ions of the isotopic group, which also contains ions with admixtures of other isotopes such as 2H, 13C, 15N, 17O, 18O, and 34S.
If peptides with molecular weights in the range between 1,000 and 5,000 atomic mass units are used for the imaging at correspondingly high mass resolution, a well-resolved isotopic group comprising several individual mass signals appears in the mass spectra of the thin-section image for every peptide. As is usual in mass spectrometry, individual mass signals of an isotopic group can immediately be summarized in the monoisotopic mass by known methods and entered in a table which corresponds to a reduced mass spectrum. It is possible to use either the monoisotopic molecular mass or the monoisotopic ion mass, which differ by the mass of one proton in the case of ionizations by MALDI. The document DE 198 03 309 C1 (C. Koster, GB 2 333 893 B; U.S. Pat. No. 6,188,064 B1, 1998) describes in detail a preferred method for the determination of the ion masses, and particularly the mass of the monoisotopic ions, which has become well known under the term “SNAP”. This method is also capable of recognizing the overlapping of isotopic groups of several peptides which differ by one or more mass units.
When the term “monoisotopic mass” is used below, it can mean either the molecular mass, i.e. the mass of the neutral molecule, or the ion mass, i.e. the mass of the protonated molecule.
The term “mass-selective image” designates an image of the tissue section which shows only the intensity distribution of the ions of this mass of a peptide, usually a monoisotopic ion mass. These images of selected masses are usually very noisy. Special types of smoothing process (see patent application DE 10 2010 009 853, for example) can be used to produce low-noise images which are very impressive and informative.
Mass spectrometry imaging is already eminently suited for classifying tissue sections according to tissue states, such as tumorous developments, and the visual representation of the tissue states. See for this the document DE 10 2004 037512 A1 (D. Suckau et al.; GB 2 418 773 B; US 2006/0063145 A1; 2004). These images are also good for measuring the distribution of pharmaceutical products of sufficient molecular dimension and their metabolites in the tissue, because the molecular weight of the pharmaceutical products and their metabolites are known and they can therefore be easily identified.
So far, however, it has only been possible in exceptional cases, and with laborious methods, to identify some of the peptides and proteins involved from such mass spectra of individual image points from thin sections, and to show, in particular, the distribution of these peptides in the thin section (see for example L. H. Cazares: “Imaging Mass Spectrometry of a Specific Fragment of Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase Kinase 2 Discriminates”, Clin Cancer Res (17), 15; 2009). The identification is of particular interest in the search for biomarkers for certain tissue states, such as cancerous tumors.
In mass spectrometry imaging, a direct identification of endogenous peptides and proteins from the thin section is so far only possible in rare cases; an identification therefore requires additional measures. In non-imaging mass spectrometry, these measures usually entail a fragmentation of the proteins or their ions to increase the information content, whether fragmentations of the protein molecules by enzymatic digest, or fragmentations of selected parent ions for the generation of daughter ions, or even combinations of both. Proteolytic peptides, measurable between 800 and 4,000 atomic mass units, are peptides which result from the enzymatic degradation of the protein chain, e.g. by digestion with the protease trypsin. Large portions of the amino acid sequence can be read from daughter ion mass spectra; this makes an identification of these proteins possible. The methods for acquiring daughter ion mass spectra consume, however, considerable quantities of substance; for mass spectrometric imaging, the amount of substance of an image point is hardly sufficient for a daughter ion spectrum acquisition. Up to now, attempts at generating daughter ion spectra showed that daughter ion spectra of moderate quality only can be obtained from one or sometimes two high-intensity peptides of an image point (see for example D. Debois et al, “MALDI-In Source Decay Applied to Mass Spectrometry Imaging: A New Tool for Protein Identification”, Analytical Chemistry, Vol. 82, 4036-45; 2009), but this is by no means sufficient for a substance identification on a larger scale.
For non-imaging MALDI mass spectrometry on individual samples it is known that the identification of proteins is particularly successful via an enzymatic, for example tryptic, digestion of the proteins in conjunction with precise mass determination of the digest peptides or acquisition of their daughter ion spectra. An excellent measurement method, known by the term “LC-MALDI” (an abbreviation for “liquid chromatography matrix-assisted laser desorption and ionization), combines a separation of the digest peptides by liquid chromatography (HPLC), preparation of MALDI samples for separated fractions and acquisition of MALDI mass spectra and daughter ion spectra by a MALDI time-of-flight mass spectrometer (see U.S. Pat. No. 7,070,949 B2; D. Suckau et al.; 2001; equivalent to GB 2 387 653 B and DE 101 58 860 B4). On the basis of the precise masses of the digest peptides and their daughter ion spectra, computer programs can be used to select proteins from large protein databases which would lead to these digest peptides and their daughter ion spectra, given known digestion and fragmentation schemes. The protein databases usually contain the sequences of the amino acids; but it is also possible to use DNA information to identify the proteins (“open reading frames”).
Some research groups have therefore already attempted to enzymatically digest the proteins of a thin tissue section in situ for a better identification of individual proteins with the aid of the digest peptides. However, this digestion leads to a strong lateral diffusion of the digest peptides, and thus to an image with far less spatial resolution. In addition, the diffusion causes a strong dilution if the digested protein was localized in a small spot only, which puts many digest peptides below the detection limit.
But even if a digestion were successful with conservation of the protein positions, one would still not have achieved the goal. Since the thin tissue sections contain complex mixtures of proteins, an extremely high mass accuracy would be required for identification. A short example might explain this: for a species whose thin section is investigated, there can easily be 50,000 known proteins in the database, and digestion of these proteins would produce millions of digest peptides. If only around half of these digest peptides, let us say half a million, fell into the favorable mass range from 800 to 4,000 atomic mass units, the digest peptide of a certain monoisotopic mass number could be one of, on average, around 150 digest peptides which could occur per atomic mass unit in the mass range between 800 and 4,000 atomic mass units. The masses of these 150 digest peptides are also relatively close together; they each have a roughly Gaussian distribution with a full width at half-maximum of around 0.25 atomic mass units. The digest peptides of the thin section could only rarely be distinguished from each other, even with maximum mass resolution and maximum mass accuracy.
Therefore, the prior art direct mass spectrometric imaging methods essentially only detect and analyze the endogenous peptides and some soluble light proteins. There is no access to the most interesting non-soluble, large or immobilized biomolecules, whether the biomolecules are immobilized by chemical preparation or by the natural structure within cells. In FFPE samples with completely cross-linked peptides and proteins, not even the endogenous peptides can be analyzed. However, if, the biomolecules of the tissue section are in situ enzymatically digested, the vast and complex mixture of digest products in each image point and the limited mass accuracy does not allow the identification of the biomolecules by the usual identification procedures.